Use of multiple sequencing technologies to produce a high-quality genome of the fungus Pseudogymnoascus destructans, the causative agent of bat White-Nose Syndrome


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Title:
Use of multiple sequencing technologies to produce a high-quality genome of the fungus Pseudogymnoascus destructans, the causative agent of bat White-Nose Syndrome
Series Title:
Microbiology resource announcements
Creator:
Drees, Kevin P.
Palmer, Jonathan M.
Sebra, Robert
Lorch, Jeffrey M.
Chen, Cynthia
Wu, Cheng-Cang
Bok, Jin Woo
Keller, Nancy P.
Blehert, David S.
Cuomo, Christina A.
Lindner, Daniel L.
Jeffrey T. Foster
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American Society for Microbiology
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1 online resource

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White-nose syndrome ( lcsh )
Bats ( lcsh )
Genomes ( lcsh )
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serial ( sobekcm )

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Abstract:
White-nose syndrome has recently emerged as one of the most devastating wildlife diseases recorded, causing widespread mortality in numerous bat species throughout eastern North America. Here, we present an improved reference genome of the fungal pathogen Pseudogymnoascus destructans for use in comparative genomic studies.
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2 p.

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University of South Florida
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University of South Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
K26-05623 ( USFLDC DOI )
k26-5623 ( USFLDC Handle )

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University of South Florida
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UseofMultipleSequencingTechnologiesToProduceaHigh-Quality GenomeoftheFungus Pseudogymnoascusdestructans,theCausative AgentofBatWhite-NoseSyndrome KevinP.Drees,a JonathanM.Palmer,bRobertSebra,cJeffreyM.Lorch,dCynthiaChen,eCheng-CangWu,eJinWooBok,fNancyP.Keller,fDavidS.Blehert,d ChristinaA.Cuomo,gDanielL.Lindner,b JeffreyT.FosteraDepartmentofMolecular,CellularandBiomedicalSciences,UniversityofNewHampshire,Durham,NewHampshire,USAa;CenterforForestMycologyResearch, NorthernResearchStation,U.S.ForestService,Madison,Wisconsin,USAb;DepartmentofGeneticsandGenomicSciencesandIcahnInstituteforGenomicsandMultiscale Biology,IcahnSchoolofMedicineatMountSinai,NewYork,USAc;U.S.GeologicalSurvey,NationalWildlifeHealthCenter,Madison,Wisconsin,USAd;IntactGenomics, SaintLouis,Missouri,USAe;DepartmentofBacteriologyandDepartmentofMedicalMicrobiologyandImmunology,UniversityofWisconsin,Madison,Wisconsin,USAf; GenomeSequencingandAnalysisProgram,BroadInstituteofMITandHarvard,Cambridge,Massachusetts,USAgWhite-nosesyndromehasrecentlyemergedasoneofthemostdevastatingwildlifediseasesrecorded,causingwidespreadmortalityinnumerousbatspeciesthroughouteasternNorthAmerica.Here,wepresentanimprovedreferencegenomeofthefungal pathogen Pseudogymnoascusdestructans foruseincomparativegenomicstudies. Received 11April2016 Accepted 18May2016 Published 30June2016 Citation DreesKP,PalmerJM,SebraR,LorchJM,ChenC,WuC-C,BokJW,KellerNP,BlehertDS,CuomoCA,LindnerDL,FosterJT.2016.Useofmultiplesequencing technologiestoproduceahigh-qualitygenomeofthefungus Pseudogymnoascusdestructans,thecausativeagentofbatwhite-nosesyndrome.GenomeAnnounc4(3): e00445-16.doi:10.1128/genomeA.00445-16. Copyright 2016Dreesetal.Thisisanopen-accessarticledistributedunderthetermsofthe CreativeCommonsAttribution4.0Internationallicense. Address correspondencetoJeffreyT.Foster,jeff.foster@unh.edu. Sinceemergingin2006,white-nosesyndromehasrapidly spreadacrosseasternNorthAmericafromanindexsitein NewYork,causingthemortalityofmillionsofbatsofnumerous species(1).Thepathogenwasidentiedas Pseudogymnoascus destructans (2, 3),apsychrophilicfungusthatinfectsbatsduring hibernation, whenbatbodytemperaturesdecreasetotheambient temperatureoftheirhibernacula( 4).Thegrowthofthisfunguson bats andsubsequentinvasionofepidermaltissuescancausea cascadeofdeleteriousphysiologicalchangesresultinginhigh mortalityrates(5, 6).ThefungusiswidespreadinEurasia( 7), and initialgeneticandexperimentalresultssuggestthatitwas recentlyintroducedtoNorthAmerica( 10, 11).Criticallymissing, however, aredetailedgenomicanalysesof P.destructans toshed lightontheorigins,evolution,globaldispersal,andpathogenicity ofthisfungus. Initialgenomesequencingeffortsfor P.destructans used454and Illuminaplatforms;however,unresolvedrepeatregionsinthegenomeresultedinfragmenteddraftassemblies(12).Therefore,togenerate ahigh-qualitycontiguousassemblyofthe P.destructans genome,weutilizedacombinationofdatafromPacBioSMRTreads, IlluminaMiSeq250-bppaired-endreads(SRR1952982),8-kb454 “jumping”libraries(SRP001346),andendsequencesfromanunbiasedrandomshearbacterialarticialchromosome(BAC)library withanaverageinsertsizeof100kb( 13). The PacBioreadswereassembledwiththePacBioSMRTversion2.2analysispipeline(14)andyielded153contigs.UsingIllumina 250-bppaired-endMiSeqreadsquality-trimmedtoQ30 withea-utilsversion1.1.2(15)anderror-correctedwithHammer (16),4548-kbjumpingreads,and 100-kb BAClibraryendsequences,thecontigswereextendedandscaffoldedwithSSPACE version3.0(17);gapswereremovedwithGapFillerversion1.10 (18).TwentyiterationsofSSPACE/GapFillerwerefollowedby Pilon version1.8(19)tocorrectlocalmisalignmentsandindels from theassembly,resultingin96scaffolds.Aself-queryofthe improvedscaffoldswiththeNUCmerapplicationofMUMmer version3.23(20)indicatedthatthesmallerscaffolds( 42,633 bp) andafewlarger scaffoldswerecompleteornearlycompleteduplicationsofthesequenceinotherscaffolds.Thirteenscaffolds,which hada 95%identityover95%oftheirlengthwithalargerscaffoldin theassembly,wereremoved.Thenalassemblycontains83scaffolds, is35.818201Mbinsize,hasan N50valueof1.168637Mb,andcontainsonly7,812bpofgaps.Thisisasignicantimprovementcomparedtopreviousdraftassembliesof P.destructans (AEFC01:1,847 scaffolds,30.6849Mb, N50105.158kb,2,328,879bpNs;AYKP01: 5,304scaffolds,30.2827Mb, N5017.914kb,0Ns). Thenew P.destructans genomecontains38.17%repetitivesequenceelements(RepeatModelerversion1.08,RepeatMasker version4.05, http://www.repeatmasker.org),whichrequireda combination oflong-readandlarge-insertsequencingtechnologiestoscaffold.Thenearlycompleteassemblypresentedhere shouldserveasavaluableresourcetofacilitatefungaldiseaseresearch. Nucleotidesequenceaccessionnumbers. Thiswhole-genome shotgunprojecthasbeendepositedinDDBJ/ENA/GenBankundertheaccessionnumber LAJJ00000000.Theversiondescribedin this paperistherstversion,LAJJ01000000.ACKNOWLEDGMENTSWewouldliketothankKatyPariseforpreparingtheDNAsequencedin thisstudy.Useoftrade,product,orrmnamesisfordescriptivepurposes onlyanddoesnotimplyendorsementbytheU.S.Government. crossmark GenomeAnnouncements May/June2016Volume4Issue3e00445-16 genomea.asm.org 1 on February 5, 2020 at Univ of South Florida http://mra.asm.org/ Downloaded from

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FUNDINGINFORMATIONThiswork,includingtheeffortsofJeffreyT.Foster,wasfundedby NationalScienceFoundation(NSF)(DEB-1336290).Thiswork, includingtheeffortsofJeffreyT.Foster,wasfundedbyDOI|U.S. GeologicalSurvey(USGS).Thiswork,includingtheeffortsofKevin P.Drees,JonathanM.Palmer,DanielLindner,andJeffreyT.Foster, wasfundedbyDOI|U.S.FishandWildlifeService(USFWS).REFERENCES1. TurnerGG,ReederDM,ColemanJH.2011.Ave-yearassessmentof mortalityandgeographicspreadofwhite-nosesyndromeinNorthAmericanbatsandalooktothefuture.BatResNews 52:13. 2. LorchJM,MeteyerCU,BehrMJ,BoylesJG,CryanPM,HicksAC, BallmannAE,ColemanJTH,RedellDN,ReederDM,BlehertDS.2011. Experimentalinfectionofbatswith Geomycesdestructans causeswhitenosesyndrome.Nature 480:376. http://dx.doi.org/10.1038/ nature10590. 3. Blehert DS,HicksAC,BehrM,MeteyerCU,Berlowski-ZierBM, BucklesEL,ColemanJTH,DarlingSR,GargasA,NiverR,Okoniewski JC,RuddRJ,StoneWB.2009.Batwhite-nosesyndrome:anemerging fungalpathogen?Science 323:227. http://dx.doi.org/10.1126/ science.1163874. 4. Verant ML,BoylesJG,WaldrepW,WibbeltG,BlehertDS.2012. Temperature-dependentgrowthof Geomycesdestructans,thefungusthat causesbatwhite-nosesyndrome.PLoSOne 7:e46280. http://dx.doi.org/ 10.1371/journal.pone.0046280. 5. Cryan PM,MeteyerCU,BoylesJG,BlehertDS.2010.Wingpathologyof white-nosesyndromeinbatssuggestslife-threateningdisruptionofphysiology.BMCBiol 8:135. http://dx.doi.org/10.1186/1741-7007-8-135. 6. Verant ML,MeteyerCU,SpeakmanJR,CryanPM,LorchJM,Blehert DS.2014.White-nosesyndromeinitiatesacascadeofphysiologicdisturbancesinthehibernatingbathost.BMCPhysiol 14:10. http://dx.doi.org/ 10.1186/s12899-014-0010-4. 7. Hoyt JR,SunK,PariseKL,LuG,LangwigKE,JiangT,YangS,Frick WF,KilpatrickAM,FosterJT,FengJ.2015.Widespreadbatwhite-nose syndromefungus,northeasternChina.EmergInfectDis 22:140. http://dx.doi.org/10.3201/eid2201.151314. 8. Puechmaille SJ,FrickWF,KunzTH,RaceyPA,VoigtCC,WibbeltG, TeelingEC.2011.White-nosesyndrome:isthisemergingdiseaseathreat toEuropeanbats?TrendsEcolEvol 26:570. http://dx.doi.org/ 10.1016/j.tree.2011.06.013. 9. Zukal J,BandouchovaH,BrichtaJ,CmokovaA,JaronKS,KolarikM, KovacovaV,KubtovA,NovkovA,OrlovO,PikulaJ,PresetnikP, ubaJ,ZahradnkovA,Jr,MartnkovN.2016.White-nosesyndrome withoutborders: Pseudogymnoascusdestructans infectiontoleratedinEuropeandpalearcticAsiabutnotinNorthAmerica.SciRep 6:19829. http:// dx.doi.org/10.1038/srep19829. 10. Leopardi S,BlakeD,PuechmailleSJ.2015.White-nosesyndromefungus introducedfromEuropetoNorthAmerica.CurrBiol 25:R217–R219. http://dx.doi.org/10.1016/j.cub.2015.01.047. 11. Warnecke L,TurnerJM,BollingerTK,LorchJM,MisraV,CryanPM, WibbeltG,BlehertDS,WillisCKR.2012.Inoculationofbatswith European Geomycesdestructans supportsthenovelpathogenhypothesis fortheoriginofwhite-nosesyndrome.ProcNatlAcadSciUSA 109: 6999. http://dx.doi.org/10.1073/pnas.1200374109. 12. Chibucos MC,CrabtreeJ,NagarajS,ChaturvediS,ChaturvediV.2013. Draftgenomesequencesofhumanpathogenicfungus Geomycespannorumsensulato andbatwhitenosesyndromepathogen Geomyces (Pseudogymnoascus) destructans.GenomeAnnounc 1 (6):e01045-13. http:// dx.doi.org/10.1128/genomeA.01045-13. 13. Bok JW,YeR,ClevengerKD,MeadD,WagnerM,KrerowiczA, AlbrightJC,GoeringAW,ThomasPM,KelleherNL,KellerNP,Wu CC.2015.Fungalarticialchromosomesforminingofthefungalsecondarymetabolome.BMCGenomics 16:343. http://dx.doi.org/10.1186/ s12864-015-1561-x. 14. Chin CS,AlexanderDH,MarksP,KlammerAA,DrakeJ,HeinerC, ClumA,CopelandA,HuddlestonJ,EichlerEE,TurnerSW,KorlachJ . 2013.Nonhybrid,nishedmicrobialgenomeassembliesfromlong-read SMRTsequencingdata.NatMethods 10:563. http://dx.doi.org/ 10.1038/nmeth.2474. 15. Aronesty E.2013.Comparisonofsequencingutilityprograms.Open BioinformaJ 7:1–8. http://dx.doi.org/10.2174/1875036201307010001. 16. Medvedev P,ScottE,KakaradovB,PevznerP.2011.Errorcorrectionof high-throughputsequencingdatasetswithnon-uniformcoverage.Bioinformatics 27:i137–i141. http://dx.doi.org/10.1093/bioinformatics/btr208. 17. Boetzer M,HenkelCV,JansenHJ,ButlerD,PirovanoW.2011.Scaffoldingpre-assembledcontigsusingSSPACE.Bioinformatics 27: 578. http://dx.doi.org/10.1093/bioinformatics/btq683. 18. Nadalin F,VezziF,PolicritiA.2012.GapFiller:adenovoassembly approachtollthegapwithinpairedreads.BMCBioinformatics 13(suppl 1):S8. http://dx.doi.org/10.1186/1471-2105-13-S14-S8. 19. Walker BJ,AbeelT,SheaT,PriestM,AbouellielA,SakthikumarS, CuomoCA,ZengQ,WortmanJ,YoungSK,EarlAM.2014.Pilon:an integratedtoolforcomprehensivemicrobialvariantdetectionandgenomeassemblyimprovement.PLoSOne 9:e112963. http://dx.doi.org/ 10.1371/journal.pone.0112963. 20. Kurtz S,PhillippyA,DelcherAL,SmootM,ShumwayM,AntonescuC, SalzbergSL.2004.Versatileandopensoftwareforcomparinglargegenomes.GenomeBiol 5:R12. http://dx.doi.org/10.1186/gb-2004-5-2-r12.Dreesetal. GenomeAnnouncements 2 genomea.asm.org May/June2016Volume4Issue3e00445-16 on February 5, 2020 at Univ of South Florida http://mra.asm.org/ Downloaded from


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